Nucleic acid encoding truncated integrins

ABSTRACT

tr-αllb, a soluble, truncated integrin found to be exclusively expressed in tumor cells is provided. An additional truncated integrin, tr-β3, has also been found to be exclusively expressed in tumor cells. Diagnostic compositions including nucleic acid probes and antibodies and methods for detecting the presence of tr-αllb and tr-β3 to identify the presence of tumor cells in a sample are also provided.

SPONSORSHIP

Work on this invention was supported in part by the National Institutesof Health Grant Nos. RO1-CA47115 and CA69845. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to truncated integrins and moreparticularly, an isolated, soluble, truncated integrin, exclusivelyexpressed in tumor cells.

BACKGROUND OF THE INVENTION

Tumor cell metastasis is a complex multistep process involving homotypicand heterotypic interactions among tumor cells and host cells (i.e.,platelets, endothelial cells, etc.), in addition to tumor cellinteractions with the extracellular matrix. These interactions aremediated by a variety of cell surface receptors including cadherins,selecting, and integrins. Tang, D. G. et al., Invasion Metastasis95:109-122 (1994).

Integrins are heterotypic adhesion receptors involved in cell-cell andcell-matrix interactions. At least 15 alpha (α) and 8 beta (β) subunitshave been identified that can pair differently to form more than 20receptors. Hynes, R. O., Cell 69:11-25 (1992); Schwartz, M. A., et al.,Ann. Rev. Cell Dev. Biol. 11:549-599 (1995). Most cDNAs coding for theknown integrins have been cloned and sequenced, and their geneslocalized to chromosomes. (Block, K. L. et al., Stem Cells 13:135-145(1995).

The platelet integrin αllbβ3 (also known as GP llb-llla) is theprototypical integrin receptor and its structure has been studied ingreat detail. Calvete, J. J., Throm. Haemostasis 72:1-15 (1994).Integrins generally contain a large extracellular domain formed by the α(˜1,000 amino acids) and β (˜750 amino acids) subunit, a singletransmembrane segment from each subunit, and two short cytoplasmictails, with the exception of β4, whose cytoplasmic tail is more than1,000 amino acid residues in length. Sastry, S. K. et al., Current Opin.Cell Biol. 5:819-831 (1993). The β subunit is a single chain polypeptidewith 4 cysteine repeats in the extracellular domain. The α subunit is asingle gene product that is postranslationally cleaved into the lightand heavy chain which are re-connected by a disulfide bond. The lightchain of the α subunit contains the transmembrane and the cytoplasmictail. Even though integrins were originally thought to function purelyas anchor molecules, they are also known as signaling receptors.Integrin cytoplasmic tails do not have intrinsic enzymatic activity, butby recruiting and activating tyrosine (pp125FAK, pp60src), serine (PKCa,ERK, JNK, ILK) or lipid (cPLA2, PI3K, PI4P5K) kinases, they cansimultaneously control multiple signaling pathways such as the MAPkinase and JAK-TAT pathways. Clark, E. A. et al., Science 268:233-2239(1995); Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599(1995).

Human and rat cDNAs of αllb as well as human cDNA of β3 have beencloned. Poncz, M. et al., J. Biol. Chem. 262:8476-8482 (1987); Poncz, M.et al., Blood 75:1282-1289 (1990); Fitzgerald, L. et al., J. Biol. Chem62:3936-3939 (1987). Genes of human αllb and β3 have been localized tochromosome 17, and their structures have been determined. Heidenreich,R. et al., Biochem. 29:1232-1244 (1990); Lanza, F. et al., J. Biol.Chem. 265:18098-18103 (1990); Bray, P. F. et al., J Clin. Invest.80:1812-1817 (1987); Sosnoski, D. M. et al., J. Clin. Invest.81:1993-1998 (1988); Rosa, J. P. et al., Blood 72:593-600 (1988). Theintegrin αllb gene is believed to be under stringent megakaryocytespecific transcriptional control whereas β3 is widely expressed.Prandini, M. H. et al., J. Biol. Chem. 267:10370-10374 (1992); Calvete,J. J. et al., Throm. Haemostasis 72:1-15 (1994); Block, K. L. et al.,Stem Cells 13:135-145 (1995).

Integrins are conformationally labile and they can exist in an inactiveand 20 active form. The inactive integrin recognizes ligand with lowaffinity while the active integrin recognizes ligand with high affinity.For example, in resting platelets αllbβ3 is constitutively expressed inan inactive form, and only after platelet activation is the integrinconverted to an active state which then binds plasma fibrinogen withhigh affinity, thereby resulting in platelet aggregation. Shattil, S. J.et al., Current Opin. Cell Biology 6:695-704 (1994). Integrins can beactivated by extracellular signals such as divalent cations (Mn²⁺, Ca²⁺)or by treatment with certain activating mAbs. Schwartz, M. A. et al.,Annu. Rev. Cell Dev. Biol. 11:549-599 (1995). Such activation induces aconformational change without involving cellular metabolism, aphenomenon referred to as "outside-in" signaling. On the other hand,growth factor-mediated activation of intracellular kinases andphosphatases can result in integrin activation, a phenomenon referred toas "inside-out" signaling. Integrins therefore participate inbi-directional signaling. Schwartz, M. A. et al., Annu. Rev. Cell Dev.Biol. 11:549-599 (1995). Several reports in the literature indicate thatinside-out signaling is mediated by the cytoplasmic tail of the αsubunit, and outside-in signaling by the cytoplasmic tail of the βsubunit. Schwartz, M. A. et al., Annu. Rev. Cell Dev. Biol. 11:549-599(1995); Shattil, S. J. et al., Current Opin. Cell Biology 6:695-704(1994).

Although the relationship between integrin receptors and metastasis isvariable, most integrins expressed by tumor cells are also expressed bytheir normal counterparts, however some tumor cells express certainintegrins that are not expressed by their normal counterparts, aphenomenon referred to as "ectopic" or "abnormal" expression. Forexample, several groups have demonstrated the ectopic expression ofαllbβ3 in non-megakaryocytic cells derived from solid tumors. Chen, Y.Q. et al., J. Biol. Chem. 267:17314-17320 (1992); Trikha, M. et al.,Cancer Res. 56:5071-5078 (1996); Trikha, M. et al., Cancer Res.57:2522-2528 (1997); Boekerche, H. et al., Blood 74:658-663 (1989);Kamiyama, M. et al., Cancer Res. 53:221-223 (1993); Chiang, H. S. etal., Biochem. et Biophys. Acta 1224:506-516 (1994); Puerschel, W. Ch. etal., British J. Dermatol. 135:883-887 (1996). This receptor participatesin tumor cell-platelet, -endothelial cell, and -ECM interactions.Chiang, H. S. et al., Biochim. et Biophys. Acta 1224:506-516 (1996);Tang, D. G. et al., Invasion Metastasis 95:109-122 (1994); Honn, K. V.et al., Exp. Cell Res. 201:23-32 (1992). In addition, subpopulationsfrom melanoma tumors, which differ in their metastatic potential,demonstrate a positive correlation between αllbβ3 expression and lungcolony formation. Tang, D. G. et al., Intl. J. Cancer 54:338-347 (1993).Two mAbs directed to αllbβ3, 10E5 and PAC-1, inhibit lung colonizationof tail vein injected human prostate adenocarcinoma DU-145 cells in SCIDmice. Trikha, M. et al., Prostate In Press (1997). Collectively, thesefindings suggest that the platelet αllbβ3 integrin is ectopicallyexpressed in non-megakaryocytic lineage tumor cells, and it participatesin tumor cell metastasis. It is conceivable that as a result oftransformation events transcription of the αllb gene is elevated in sometumors which allows them to interact with the host in a platelet-typemanner thereby facilitating the metastatic process. Recent studiesindicate that human melanoma cells express an intracellular pool ofconstitutively active αllbβ3, because these permeabilized cellsrecognize PAC-1, a unique mAb because it specifically recognizes thehigh affinity state of αllbβ3 integrin. Shattil, S. J. et al., J. Biol.Chem. 260:11107-11114 (1995); Trikha, M. et al., Cancer Res.57:2522-2528 (1997). Earlier reports in the literature indicate that CHOcells transfected with wt-αllb and wt-β3 cDNAs constitutively expressαllbβ3 in a low affinity state. Schwartz, M. A. et al., Annu. Rev. CellDev. Biol. 11:549-599 (1995); O'Toole, T. E. et al., J. Cell Biol.124:1047-1059 (1994). However, transfection with mutant αllb constructsthat either have a point mutation in the cytoplasmic tail or completedeletion of the tail, result in constitutive expression of the integrinin a high affinity state. Schwartz, M. A. et al., Annu. Rev. Cell Dev.Biol. 11:549-599 (1995); O'Toole, T. E. et al., J. Cell Biol.124:1047-1059 (1994); O'Toole, T. E. et al., Science 254:845-847 (1991).To date no such naturally occurring mutant constructs had been observed.

SUMMARY OF THE INVENTION

An isolated, truncated integrin referred to herein as tr-αllb, isprovided. tr-αllb is a novel, soluble, truncated integrin found to beexclusively expressed in tumor cells. An additional truncated integrin,tr-β3, has also been found to be exclusively expressed in tumor cells.Diagnostic compositions and methods for detecting tr-αllb and tr-β3 toidentify the presence of tumor cells in a sample are also provided.

Additional objects, advantages, and features of the present inventionwill become apparent from the following description and appended claims,taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The various advantages of the present invention will become apparent toone skilled in the art by reading the following specification andsubjoined claims and by referencing the following drawings in which:

FIG. 1A is an ethidium bromide-stained agarose gel showing the productsof PCR amplification of an alternately spliced tr-αllb cDNA;

FIG. 1B is a nucleotide sequence of the 1.1 kb product of tr-αllb (SEQID NO:1);

FIG. 1C is a comparison of the partial nucleotide sequence of the 0.8 kbproduct with that of genomic αllb IVS;

FIG. 1D is a schematic representation of the predicted structuralcomparison of wt-αllb and tr-αllb;

FIG. 1E is a partial amino acid sequence of tr-αllb deduced from thecDNA (boxed amino acid sequence represents the region specific totr-αllb and the underlined amino acids represent the sequence used togenerate the antibody to tr-αllb) (SEQ ID NO:2);

FIG. 2A is an autoradiograph of the in vitro translation products forwt-αllb and tr-αllb;

FIG. 2B is an immunoblot of the in vitro translation products of wt-αllband tr-αllb probed with an antibody to tr-αllb;

FIG. 3A is an immunoblot of the total cell lysate from human melanomaWM983 and prostate adenocarcinoma DU145 cells probed with an antibody totr-αllb in the absence and presence of excess antigen;

FIG. 3B is an immunoblot of the total cell lysate from human melanomaWM983 probed with a biotinylated antibody to tr-αllb;

FIG. 3C is an immunoblot of total cellular protein from DU-145, HEL andWM-983B human carcinoma cells and normal prostate epithelial cells andplatelets. The blots were probed with antibody to tr-αllb then strippedand probed with antibody to human actin;

FIG. 3D is an immunoblot of total cellular protein from healthyvolunteers (normal) and patients with prostate adenocarcinoma (PCa)probed with antibody to tr-αllb and wt-αllb;

FIG. 4 is an immunoblot of total cell lysates from HEL and WM983B humancarcinoma cells probed with either antibody to tr-β3 or with preimmuneserum;

FIG. 5A is an immunoblot of the subcellular fractions of HEL cellsprobed with antibody to tr-αllb, wt-αllb or preimmune serum;

FIG. 5B is an immunoblot of the subcellular fractions of human melanomaWM983B cells probed with antibody to tr-αllb;

FIG. 5C is an immunoblot of the subcellular fractions of WM983B cellsprobed with an antibody to tr-β3;

FIG. 6A is a schematic representation of the structure of a wild-typeintegrin;

FIG. 6B is a schematic representation of the structure of an integrinwith wt-β3 and tr-αllb subunits;

FIG. 6C is a schematic representation of the structure of an integrinwith wt-αllb and tr-β3 subunits;

FIG. 6D is a schematic representation of the structure of a fullytruncated integrin having tr-αllb and tr-β3 as its subunits;

FIG. 7A is an immunohistogram of prostatic tumor tissue and normalprostatic tissue stained with antibody to tr-αllb;

FIG. 7B is an immunohistogram of prostatic tumor tissue and normalprostatic tissue stained with preimmune serum for anti-tr-αllb;

FIG. 7C is an immunohistogram of prostatic tumor tissue and normalprostatic tissue stained with antibody to tr-β3; and

FIG. 7D is an immunohistogram of prostatic tumor tissue and normalprostatic tissue stained with preimmune serum for anti-tr-β3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An isolated, alternately spliced variant of αllb referred to herein astr-αllb, is provided. tr-αllb contains exons 22 through 26 of wt-αllbwith exons 27 through 30 replaced with a partial sequence from intron26, but lacks the transmembrane and cytoplasmic portions of the lightchain. An antibody (referred to herein as pAb MTB1) generated to theunique sequence of tr-αllb is also provided. pAb MTB1 recognizestr-αllb, but not wt-αllb. Western blotting with pAb MTB1 identified an˜100 kDa tr-αllb protein that is expressed in human leukemia, prostateadenocarcinoma, and melanoma cells, but not in platelets or normalprostate epithelial cells. In addition, it has been found that thesemelanoma and prostate adenocarcinoma cells also express an alternatelyspliced and truncated β3 (referred to herein as tr-β3) which also lacksthe transmembrane and cytoplasmic domain. An antibody (referred toherein as pAb MTB4) generated to the unique sequence of tr-β3 is alsoprovided. Unlike wt-αllb and wt-β3, tr-αllb and tr-β3 are expressed inboth cytoplasmic and membrane compartments.

The nucleic acid sequence of the cDNA encoding tr-αllb and its deducedamino acid sequence are set forth in SEQ ID NOS: 1 and 2, respectively.In a preferred embodiment, the isolated nucleic acid molecule of theinvention comprises the nucleotide sequence of SEQ ID NO: 1, orhomologues therefore. In another preferred embodiment, the isolated andpurified polypeptide of the invention comprises the amino acid sequenceof SEQ ID NO: 2, as well as biological equivalents.

In another embodiment, the isolated and purified nucleic acid moleculeof the present invention is incorporated into an appropriate recombinantexpression vector, e.g., viral or plasmid, which is capable oftransfecting an appropriate host cell, either eukaryotic (e.g.,mammalian) or prokaryotic (e.g., E. coli). Such nucleic acid moleculesmay involve alternate nucleic acid forms, such as cDNA, gDNA (genomicDNA), and DNA prepared by partial or total chemical synthesis. Thenucleic acid molecule may also be accompanied by additional regulatoryelements, such as promoters, operators and regulators, which arenecessary and/or may enhance the expression of the protein encoded bythe nucleic acid molecule. It is further contemplated that thenucleotide sequence of the present invention may be utilized tomanufacture tr-αllb using standard synthetic methods. Various expressionvectors and methods for introducing such vectors into cells are known inthe art and are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989).

In yet another embodiment, the present invention provides a recombinanthost cell transfected with a vector of the present invention comprisingthe nucleic acid molecule of the present invention. Preferably, arecombinant host cell comprises a polynucleotide under thetranscriptional control of regulatory signals functional in therecombinant host cell, wherein the regulatory signals appropriatelycontrol expression of the polynucleotide of the present invention in amanner to enable all necessary transcriptional and post-transcriptionalmodifications. The incorporation of these sequences into prokaryotic andeukaryotic host cells by standard transformation and transfectionprocesses, thus provides for the production of encoded tr-αllb protein.

In another aspect, DNA sequence information provided by the presentinvention allows for the preparation of relatively short DNA (or RNA)sequences or probes that are identical to or hybridize to the nucleotidesequence disclosed herein. Nucleic acid probes of an appropriate lengthare prepared based on a consideration of the nucleotide sequence of SEQID NO: 1 or to the nucleotide sequence of tr-β3 wherein said sequence isset forth in Fitzgerald, L. A. et al., J. Biol. Chem. 262:3936-3939(1987) and Djaffar, I. et al., Biochem. J. 300:69-74 (1994) herein,expressly incorporated by reference. Fitzgerald et al. teaches thecomplete wild-type sequence of β3 (also known as glycoprotein IIIa) andDjaffar et al. discloses the 340 base pair insert at position 1281 ofthe wild-type sequence which results in tr-β3 integrin. The probes canbe used in a variety of assays appreciated by those skilled in the art,for detecting the presence of complementary sequences in a given sample.The probes may be useful in prognostic and diagnostic applications.

A preferred nucleic acid sequence employed for hybridization studies orassays includes probe molecules that are complementary to at least a 10to 70 or so long nucleotide stretch of the polynucleotide sequence shownin SEQ ID NO: 1 or to the nucleotide sequence of tr-β3. A size of atleast 10 nucleotides in length helps to ensure that the fragment will beof sufficient length to form a duplex molecule that is both stable andselective. Molecules having complementary sequences over stretchesgreater than 10 bases in length are generally preferred in order toincrease stability and selectivity of the hybrid, and thereby improvethe quality and degree of specific hybrid molecules obtained. It will beappreciated that nucleic acid molecules having gene-complementarystretches of 25 to 40 nucleotides, 55 to 70 nucleotides, or even longerwhere desired, may be preferred. Such fragments can be readily preparedby, for example, directly synthesizing the fragment by chemical means,by application of nucleic acid reproduction technology, such as the PCRtechnology of U.S. Pat. No. 4,683,202, or by excising selected DNAfragments from recombinant plasmids containing appropriate inserts andsuitable restriction enzyme sites. In certain embodiments, it is alsoadvantageous to use oligonucleotide primers. The sequence of suchprimers is designed using the polynucleotide of the present inventionand is used with PCR technology.

In one embodiment, the present invention provides an antibodyimmunoreactive with the tr-αllb polypeptide. In another embodiment, theinvention provides an antibody immunoreactive with the tr-β3polypeptide. In a preferred embodiment, polyclonal antibody pAb MTB1 isprovided, which is specific for tr-αllb. In another preferred embodimentpolyclonal antibody pAb MTB4, specific for tr-β3, is provided. Alsocontemplated by the present invention are antibodies immunoreactive withhomologues or biologically equivalent polynucleotides and polypeptidesof the present invention. As used herein, the term "antibody" is used inits broadest sense to include polyclonal and monoclonal antibodies, aswell as polypeptide fragments of antibodies that retain a specificbinding activity for tr-αllb or tr-β3. One skilled in the art willappreciate that anti-tr-αllb or anti-tr-β3 antibody fragments such asFab, F(ab')₂ and Fv fragments can retain specific binding activity fortr-αllb or tr-β3 and, thus, are included within the definition of anantibody. In addition, the term "antibody" as used herein includesnaturally occurring antibodies as well as non-naturally occurringantibodies and fragments that retain binding activity. Methods of makingantibodies are known in the art. See, e.g., Harlow and Lane, Antibodies:A Laboratory Manual (Cold Spring Harbor Press, 1988).

Diagnostic methods are further provided by the present invention and maybe used to detect the presence of tr-αllb and/or tr-β3 in a sample. Inone embodiment, a method of detecting the presence of tr-αllb in asample is provided, wherein the method comprises the steps ofadministering to the sample a nucleic acid probe specific for tr-αllband detecting hybridization of the probe and nucleotide sequencesencoding tr-αllb in the sample. The methods used to detect the presenceof tr-αllb and/or tr-β3 may include, but are not limited to,amplification of the nucleic acid sequences encoding for tr-αllb and/ortr-β3 by PCR or other methods known to those skilled in the art. Inanother embodiment, a method of detecting the presence of tr-β3 in asample is provided comprising the steps of administering to the sample anucleic acid probe specific for tr-β3 and detecting hybridization of theprobe and nucleotide sequences encoding tr-β3 in the sample.Hybridization may be carried out under stringent conditions. The samplemay be any suitable biological sample including, but not limited to,tissue, blood, semen and urine.

In yet another embodiment, the present invention contemplates a processof detecting a messenger RNA transcript that encodes the polypeptide ofthe present invention, wherein the process comprises (a) hybridizing themessenger RNA transcript with a polynucleotide sequence that encodesthat polypeptide to form a duplex; and (b) detecting the duplex.Alternatively, the present invention provides a process of detecting aDNA molecule that encodes the polypeptide of the present invention,wherein the process comprises (a) hybridizing DNA molecules with apolynucleotide that encodes that polypeptide to form a duplex; and (b)detecting the duplex.

The present invention also provides methods of detecting the polypeptideof the present invention comprising the steps of immunoreacting thepolypeptide with an antibody to form an antibody-polypeptide conjugate,and detecting the conjugate, e.g., conjugating the antibodies tochemiluminescent molecules such as dioxytane-based molecules known inthe art, for use as labelled probes. Thus, methods of detecting tr-αllbprotein and/or tr-β3 protein in a sample are provided whereby antibodywhich specifically binds to tr-αllb and/or tr-β3 is administered to asample, and binding is detected. It will be appreciated by those skilledin the art that such immunoassay methods include, without limitation,radioimmunoassays, enzyme-linked immunosorbent assays, "sandwich"assays, precipitin reactions, gel diffusion immunodiffusion assays,agglutination assays and immunoelectrophoresis assays.

In yet another embodiment, the present invention provides a polypeptideor fragment thereof having the amino acid sequence of SEQ ID NO: 2,capable of binding antibodies to tr-αllb. In another embodiment, thepresent invention provides a polypeptide or fragment thereof of thetruncated integrin protein tr-β3 capable of binding antibodies specificto tr-β3. Preferably, the antibody is sequestered from a sample on asolid support. The polypeptide may comprise an indicator for conjugatedetection, e.g., a chromophore, fluorophore, biotin moeity or an enzyme.

In another aspect, the present invention contemplates a diagnostic assaykit for detecting the presence of tr-αllb and/or tr-β3 in a biologicalsample, wherein the kit comprises a first container containing a firstantibody or antibodies capable of immunoreacting with the polypeptidethat comprises tr-αllb and/or tr-β3, with the first antibody present inan amount sufficient to perform at least one assay. Preferably, an assaykit of the invention further comprises a second container containing asecond antibody that immunoreacts with the first antibody. Morepreferably, the antibodies used in an assay kit of the present inventionare monoclonal antibodies. Even more preferably, the first antibody isaffixed to a solid support. More preferably still, the first and secondantibodies comprise an indicator, and, preferably, the indicator is aradioactive label or an enzyme.

In an alternative aspect, the present invention provides a diagnosticassay kit for detecting the presence, in biological samples, of thepolynucleotides of tr-αllb and/or tr-β3. The kits comprise a containerthat contains a polynucleotide identical or complementary to a segmentof at least 10 contiguous nucleotide bases of the polynucleotide of thepresent invention.

In another embodiment, the present invention contemplates a diagnosticassay kit for detecting the presence, in a biological sample, of anantibody immunoreactive with the tr-αllb and/or tr-β3 polypeptides. Thekits comprise a container containing the polypeptide that immunoreactswith the antibody, with the polypeptide present in an amount sufficientto perform at least one assay.

It will be appreciated that the nucleic and amino acid sequences of thepresent invention can include some variation from the sequencesrepresented by and complementary to the sequences set forth in theSequence Listing but must be substantially represented by orcomplementary to those set forth therein. By "substantially representedby" or "substantially complementary to" is meant that any variationtherein does not impair the functionality of the sequence to anysignificant degree. As used herein, the term "nucleic acid" is intendedto mean natural and synthetic linear and sequential arrays ofnucleotides and nucleosides, e.g. in cDNA, genomic DNA (gDNA), mRNA, andRNA, oligonucleotides, oligonucleosides and derivatives thereof. It willalso be appreciated that such nucleic acids can be incorporated intoother nucleic acid chains referred to as "vectors" by recombinant-DNAtechniques such as cleavage and ligation procedures. The terms"fragment" and "segment" are as used herein with reference to nucleicacids (e.g., cDNA, genomic DNA, i.e., gDNA) are used interchangeably tomean a portion of the subject nucleic acid such as constructedartificially (e.g. through chemical synthesis) or by cleaving a naturalproduct into a multiplicity of pieces (e.g. with a nuclease orendonuclease to obtain restriction fragments). As used herein, "A"represents adenine; "T" represents thymine; "G" represents guanine; "C"represents cytosine; and "U" represents uracil.

As referred to herein, the term "encoding" is intended to mean that thesubject nucleic acid may be transcribed and translated into the subjectprotein in a cell, e.g. when the subject nucleic acid is linked toappropriate control sequences such as promoter and enhancer elements ina suitable vector (e.g. an expression vector) and the vector isintroduced into a cell. The term "polypeptide" is used to mean three ormore amino acids linked in a serial array.

As referred to herein, the term "capable of hybridizing under highstringency conditions" means annealing a strand of DNA complementary tothe DNA of interest under highly stringent conditions. Likewise,"capable of hybridizing under low stringency conditions" refers toannealing a strand of DNA complementary to the DNA of interest under lowstringency conditions. In the present invention, hybridizing undereither high or low stringency conditions would involve hybridizing anucleic acid sequence (e.g., the complementary sequence to SEQ ID NO: 1or portion thereof), with a second target nucleic acid sequence. "Highstringency conditions" for the annealing process may involve, forexample, high temperature and/or low salt content, which disfavorhydrogen bonding contacts among mismatched base pairs. "Low stringencyconditions" would involve lower temperature, and/or lower saltconcentration than that of high stringency conditions. Such conditionsallow for two DNA strands to anneal if substantial, though not nearcomplete complementarity exists between the two strands, as is the caseamong DNA strands that code for the same protein but differ in sequencedue to the degeneracy of the genetic code. Appropriate stringencyconditions which promote DNA hybridization, for example, 6×SSC at about45° C., followed by a wash of 2×SSC at 50° C. are known to those skilledin the art or can be found in Current Protocols in Molecular Biology,John Wiley & Sons, NY (1989), 6.31-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C. Inaddition, the temperature in the wash step can be increased from lowstringency at room temperature, about 22° C., to high stringencyconditions, at about 75° C. Other stringency parameters are described inManiatis, T., et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring N.Y., (1982), at pp.387-389; see also Sambrook J. et al., Molecular Cloning: A LaboratoryManual, Second Edition, Volume 2, Cold Spring Harbor Laboratory Press,Cold Spring, N.Y. at pp. 8.46-8.47 (1989).

As used herein, the term "specifically binds" refers to a non-randombinding reaction between two molecules, for example between an antibodymolecule immunoreacting with an antigen.

SPECIFIC EXAMPLE 1 Results

Detection of wt-αllb and tr-αllb integrin cDNA. To conclusivelydemonstrate that platelet αllb integrin is transcribed innon-megakaryocytic tumor cells, the full length αllb cDNA from celllines derived from solid tumors was sequenced. The 5'-region of the αllbcDNA was amplified by 5'-RACE and published in Trikha, M. et al.,Prostate (In Press, 1997). The remaining 1.1 kb of the αllb cDNA wasamplified by 3'-RACE. In addition to the expected 1.1 kb fragment, thereis a novel PCR fragment of ˜0.8 kb expressed in human prostateadenocarcinoma, leukemia, and melanoma cells (FIG. 1). The PCR productsresult from reverse transcribed poly A RNA and not contaminating genomicDNA based on the following reasons. First, RNA treated with DNase I wasused for RT, second, Oligo dT primers were used for RT, third noamplification was obtained in the absence of RTase, fourth, controlreactions lacking template result in no amplification, fifth, no PCRproduct is obtained from RNA from normal cells, and sixth, no PCRproduct is obtained in the absence of a primer (FIG. 1A). The sequenceof the 1.1 kb PCR fragment, which spans exons 22 through 30 and the3'UTR, is identical to wt-αllb (2,288 bp-3,333 bp; Frachet, P. et al.,Mol. Biol. Rep. 14:27-33 (1990)). Sequencing of the ˜0.8 kb PCR productrevealed it to be an alternately spliced product containing exons 22through 26, with exons 27 through 30 replaced by a partial sequence fromintron 26 (11,000 bp-11,243 bp; Heidenreich, R. et al., Biochemistry29:1232-1244 (1990)) and the poly A tail (FIG. 1C). A schematicrepresentation of the structure of wt-αllb (1.1 kb) and alternatelyspliced tr-αllb (˜0.8 kb) is shown in FIG. 1D.

Northern blotting of human prostate, leukemia, and melanoma mRNA withthe alternately spliced tr-αllb cDNA as a probe (data not shown) or witha wt-αllb probe (Trikha, M. et al, Cancer Res. 57:2522-2528 (1997);Trikha, M. et al., Prostate In Press (1997)) reveals a single band thathas a similar size as platelet αllb mRNA. This result is attributed tothe ˜300 bp difference in molecular mass between wt-αllb and tr-αllbmRNA that cannot be resolved in this region of the gel. Altogether,these findings suggest that wt-αllb and tr-αllb are similar with theexception of exons 27-30.

Methods

3'-Rapid Amplification of cDNA ends (3'-RACE). Total RNA from tumorcells was obtained by the acid guanidinium isothiocyanate method usingthe Tri-Reagent kit (Molecular Research Center, Inc., Cincinnati, Ohio).To remove contaminating DNA, total RNA (2 μg) was treated with RNasefree DNase I (Life Technologies, Gathersburg, Md.) in PCR buffer (20 mMTris-HCl, pH 8.4, 50 mM KCl, 2.5 mM MgCl₂), and DNase I was neutralizedwith EDTA (1.2 mM, 65° C. for 15 min). RNA was reverse transcribed andamplified by using the 3'-RACE kit (Life Technologies). Briefly, cDNAsynthesis was initiated by the addition of cDNA synthesis adaptor primer(AP: 5'-GGC CAC GCG TCG ACT AGT ACT TTT TTT TTT TTT TTT T-3', SEQ IDNO:3), and Superscript II for 50 min at 42° C. The reaction wasterminated by incubation at 70° C. for 15 min followed by treatment withRNase H at 37° C. for 20 min. The RT mixture was amplified in a totalvolume of 50 μl of PCR buffer containing 250 ng of αllb gene specificsense primer (5'-CTG GM GAG GCT GGG AGT C-3', SEQ ID NO:4), 200 nM ofAbridged universal amplification primer (AUAP: 5'-GGC CAC GCG TCG ACTAGT AC-3', SEQ ID NO:5), 200 μM each of dATP, dCTP, dGTP, and dTTP,TaqStart antibody (Clontech, Palo Alto, Calif.), and Amplitaq DNApolymerase (Perkin-Elmer Cetus, Foster City, Calif.). PCR was performedin a GenAmp PCR system 9600 (Perkin-Elmer Cetus) by using the followingconditions: one cycle of 94° C. for 10 s; thirty cycles of 94° C. for20s, 55° C. 1 min, and 72° C. for 2 min, and one cycle of 72° C. for 7min. An aliquot (1 μl) of the first round PCR product was reamplifiedunder the exact conditions mentioned above with the substitution of theαllb sense primer with a nested-αllb sense primer (5'-GCA GAT ACG GAGCAA GAA CA-3', SEQ ID NO:6) and AUAP. PCR products were separated on a1% agarose-TBE gel and visualized by ethidium bromide staining.

DNA sequencing. PCR products were separated on a 1% agarose-TAE gel andpurified with the Qiagen kit (Chatsworth, Calif.). PCR products weredirectly sequenced by the Taq dyedeoxynucleotide Terminator CycleSequencing kit from Applied Biosystems (Foster City, Calif.) byemploying the standard protocol on a Perkin Elmer thermal cycler (model480). Sequencing reactions were run on a DNA sequencer (ABI, model 373),and the entire PCR product was sequenced by primer walking.Additionally, the PCR products were cloned into the pCR2.1 TOPO vector(Invitrogen, La Jolla, Calif.) and sequenced with T-7 and SP-6 primers.Sequencing was confirmed from several clones and different PCR products.The sequence was aligned to αllb cDNA (Genbank accession number M34480;Frachet, P. et al., Mol. Biol. Rep. 14:27-33 (1990)), and genomic αllb(Genbank accession number M33320; Heidenreich, R. et al., Biochemistry29:1232-1244 (1990)) by using the MacVector™ 4.1 software on a MacintoshQuadra 630 computer.

SPECIFIC EXAMPLE 2 Results

Predicted structure of tr-αllb. Theoretical translation of the alternatetr-αllb product indicates that the protein contains only 7 amino acidsfrom the light chain (151-157, FIG. 1E), thus it does not contain atransmembrane or cytoplasmic portion (FIG. 1D). Instead of the lightchain, the tr-αllb gene product contains 44 amino acids that map to αllbintervening sequence (IVS) 11,000 bp-11,131 bp (Heidenreich, R. et al.,Biochem. 29:1232-1244 (1990)), followed by a stop codon (αllb IVS:11,132 bp-11,134 bp). A peptide based on the unique sequence (171-200,FIG. 1E) that is specific to corresponding tr-αllb and αllb IVS, but isnot expressed in wt-αllb protein, was used to prepare a rabbitpolyclonal antibody (pAb MTB1). The IgG fraction of pAb MTB1 waspurified from the immunized serum by affinity chromatography on proteinG and used for Western blotting as described below.

In vitro translation of recombinant wt-αllb and tr-αllb. To demonstratethat pAb MTB1 specifically recognizes tr-αllb protein, but not wt-αllbprotein, recombinant wt- and tr-αllb proteins were produced. In vitrotranslation of wt-αllb pcDNA and tr-αllb pcDNA vectors indicated singleprotein products for each that migrated under reducing conditions atmolecular weights of ˜120 kDa and ˜100 kDa, respectively (FIG. 2A).Western blotting of the in vitro translated products with pAb MTB1demonstrated that this Ab only recognizes tr-αllb protein, but notwt-αllb protein (FIG. 2B). Altogether, these results suggest that thereduced molecular weight of wt-αllb and tr-αllb is ˜120 kDa and ˜100kDa, respectively, and pAb MTB1 recognizes tr-αllb but not wt-αllb.

Methods

Antibody preparations. A custom polyclonal antibody to tr-αllb wasprepared by Research Genetics Inc., Huntsville, Ala. The peptide (singleletter amino acid code: THGAEGMGGGRGVRVCCGPLWATLGPWEHFK, SEQ ID NO:7)was conjugated to KLH, emulsified with an equal volume of Freund'sAdjuvant and subcutaneously injected into rabbits. Primary immunizationof 0.1 mg of peptide was followed by three successive boosts of 0.1 mgeach. The third bleed with an ELISA titer for the peptide of 128,600 wasused for all experiments, and the pre-immune bleed from the same rabbitwas used as a negative control. The immunopure (G) IgG purification kit(Pierce, Rockford, Ill.) was used to purify the IgG fraction from theimmunized rabbit serum and called pAb MTB1. Protein G eluantconcentrations were monitored by Absorbance at 280 nm.

A custom antibody to tr-β3 was also prepared by Research Genetics asdescribed above for the antibody to tr-αllb with the exception of thepeptide antigen. The peptide used to prepare anti-tr-β3 antibodies hadthe amino sequence CPGASVGTGPPFFLL (single letter amino acid code, SEQID NO:8).

Biotinylated antibodies were prepared using the protein biotinylationkit from Amersham (Arlington Heights, Ill.) according to themanufacturer's instructions.

Generation of wt-αllb and tr-αllb expression vectors. A primer (5'-GCCTCT AGA GCC ACC ATG GCC AGA GCT TTG TG-3, SEQ ID NO:9) that maps to 33bp of HEL αllb cDNA (Frachet, P. et al., Mol. Biol. Rep. 14:27-33(1990)), and contains an Xbal restriction site, Kozak sequence, and anATG sequence was used in conjunction with T-7 antisense primer(Invitrogen) to PCR amplify the predicted 3.3 kb αllb insert from apBluescript vector. The gel purified PCR product was digested withXbal/Hindlll and ligated into pcDNA 3.1 (-) vector (Invitrogen). Severalclones were selected based on restriction mapping. Sequencing of thepcDNA 3.1 (-) construct revealed an insert which had complete homologyto HEL αllb cDNA from 33 bp-3,333 bp. Frachet, P. et al., Mol. Biol.Rep. 14:27-33 (1990). The tr-αllb construct was developed by using thepCR2.1 TOPO II plasmid which contained the 3'-RACE product from PC-3cells. Digestion of this plasmid by BamHl and EcoRl released an ˜300 bpfragment which was specific to tr-αllb. Meanwhile, a 2.7 kb αllbfragment was cloned into pcDNA 3.1 (+) and digested with BamHl andEcoRl. The ˜300 bp BamHI/EcoRl fragment was sub-cloned into the 2.7 kbαllb pcDNA (+) vector. Sequencing of this construct revealed arecombinant cDNA which had complete homology to wt-αllb (33 bp-2,759 bp)and contained the unique alternately spliced region specific to tr-αllb(473-753 bp refer to FIG. 1B). This construct was regarded as the fulllength tr-αllb pcDNA 3.1 (+) vector.

In vitro translation. The wt-αllb pcDNA 3.1 (-) and the tr-αllb pcDNA3.1 (+) vectors were translated in vitro by the TNT-T7 Quick coupledTranscription/Translation System (Promega, Madison, Wis.). Briefly, 1 mgof DNA template, 2 ml of [³⁵ S]methionine (1,000 Ci/mmole,) and 40 ml ofthe TNT/T7 rabbit reticulocyte lysate master mix was added to a finalvolume of 50 ml, and the reaction was incubated at 30° C. for 90 min. A10% aliquot was mixed with 2× reducing sample buffer, boiled,electrophoresed on a 7.5% SDS-PAGE gel, transferred to nitrocellulosemembrane, and the translated products were first visualized byautoradiography and then by Western blotting. The same blot was probedwith pAb MTB1 and the bound antibody detected by anti-rabbit conjugatedto HRP and ECL staining (Amersham).

SPECIFIC EXAMPLE 3 Results

Detection of tr-αllb and tr-β3 proteins in tumor cells. Earlier resultsindicate that a mAb directed to wt-αllb detects ˜120 kDa protein(reduced Mr of αllb) that is expressed in prostate adenocarcinoma cells(Trikha, M. et al., Cancer Res. 56:5071-5078 (1996)), which confirmsthat wt-αllb mRNA is translated in these cells. Thus, whether thealternately spliced tr-αllb and tr-β3 mRNA was also translated in thesetumor cells was studied. Western blotting of cell lysates from humantumor cells revealed that pAb MTB1 recognized a single band migrating at˜100 kDa (FIG. 3A) which is the Mr of in vitro translated recombinanttr-αllb protein (Specific Example 2, FIG. 2). Excess antigen (8-foldmolar excess over Ab) completely inhibited binding of pAb MTB1 (FIG.3A), indicating that the ˜100 kDa protein is immunologically related tothe antigenic sequence. These results were confirmed whenbiotinylated-pAb MTB1 also recognized the same 100 kDa protein (FIG.3B). The ˜100 kDa protein recognized by pAb MTB1 is specificallyexpressed in prostate adenocarcinoma (DU-145), melanoma (WM-983B),erythroleukemia (HEL), but not in normal prostate epithelial cells (NPE)(FIG. 3C), or platelets from three healthy volunteers, or platelets fromtwo patients with prostate adenocarcinoma (FIG. 3D). Stripping andreprobing the blot with mAb to human β-actin, and detecting the boundantibody with anti-mouse Ig conjugated to HRP and ECL staining, showedthat the same amount of cellular protein was present for all samples. Arabbit pAb was raised to tr-β3 (herein referred to as pAb MTB4), asdescribed in the Methods of Specific Example 2, and used in Westernblotting. The results, similar to those obtained with tr-αllb, indicatethat non-megakaryocytic lineage cells express a unique protein with a Mrof ˜60 kDa associated with the tr-β3 integrin protein (FIG. 4).

Since the predicted structure of tr-αllb and tr-β3 lacks thetransmembrane and cytoplasmic tail, whether these integrins could existas soluble protein was examined. Subcellular fractionation of humanerythroleukemia (HEL) and melanoma (WM 983B) cells followed by Westernblotting with pAb MTB1 and pAb MTB4 indicated that tr-αllb and tr-β3 arepresent in both cytosolic and membrane compartments (FIGS. 5A-5C). Asexpected, wt-αllb could only be detected in total cell lysate andmembrane fraction, but not in the cytosolic fraction (FIG. 5A). Thisdata clearly indicates that tr-integrins can exist as transmembranereceptors as well as soluble receptors. A model is shown in FIG. 6. Inthis model the association of the truncated subunit with the wild typesubunit would produce a membrane associated integrin. In contrast,association of tr-αllb with tr-β3 (tr-αllbβ3) would allow this complexto exist as a soluble receptor. Identification of the non-membraneassociated soluble integrins of the present invention suggests a novelform of integrin regulation. Tumor cells may therefore utilize alternatesplicing to switch an adhesion molecule such as an integrin into ananti-adhesion molecule also called disintegrin, and this phenomenon maybe critically involved during the intravasation and extravasation stepsof tumor cell dissemination.

Methods

Cells. Human prostate cancer cell lines, PC-3 and DU-145, isolated frommetastatic lesions, and human erythroleukemia cell line HEL wereobtained from American Type Culture Collection (Rockville, Md.). Humanmelanoma cell lines, WM 983A, WM 983B, and WM 35 were a gift from Dr.Meenhard Herlyn, The Wistar Institute (Philadelphia, Pa.). Normal breastepithelial cell line MCF10a was obtained from Karmanos Cancer Institute(Detroit, Mich.). These cell lines were cultured as described. Trikha,M. et al., Cancer Res. 56:5071-5078 (1996); Chen, Y. Q. et al., Int. J.Cancer 72:642-648 (1997). Normal prostate epithelial cells were a giftfrom Dr. Dharam Chopra, Wayne State University (Detroit, Mich.) or werepurchased from Clonetics (San Diego, Calif.) and cultured in Cloneticsmedium. All experiments were performed with cells that were grown toapproximately 80% confluency. Platelets from healthy volunteers orprostate adenocarcinoma patients were isolated from citrated blood asdescribed. Trikha, M. et al., Throm. Res. 73:39-52 (1994).

Subcellular fractionation of total cellular proteins. Subcellularfractionation was performed as described. Hagman, W. et al.,Prostaglandins 46:471-477 (1993). Briefly, tumor cells were washed inisotonic buffer (134 mM NaCl, 15 mM Tris-HCl, pH 7.6, 5 mM glucose, 1 mMEDTA, and 1 mM EGTA) and resuspended in homogenization buffer [25 mMTris-HCL, pH 7.6, 1 mM EGTA, aprotinin (5 μg/ml), leupeptin (10 μg/ml),1 mM PMSF]. The solution was sonicated (3×15 sec at 0° C.), centrifugedat 10,000×g for 15 min, and the resulting supernatant was recentrifugedat 100,000×g for 1 h at 4° C. The 100,000×g supernatant was removed andregarded as the cytosolic fraction. The pellet was rinsed andresuspended in homogenization buffer by sonication and regarded as themembrane fraction. Total protein was obtained by lysing cells with 20 mMTris-HCl, pH 7.6, 150 mM NaCl, 0.5% NP-40, 0.5% Tween-20, 1 mM PMSF,aprotinin (5 μg/ml), and leupeptin (10 μg/ml), and 20 mM EDTA asdescribed. Trikha, M. et al., Cancer Res. 56:5071-5078 (1996). Proteinconcentrations were determined by Bio-Rad's DC protein assay kit(Bio-Rad, Richmond, Calif.) and BCA protein Assay kit (Pierce).

Immunoblotting. Western blotting of freshly lysed solutions wasperformed as described. Trikha, M. et al., Cancer Res. 56:5071-5078(1996). Briefly, cellular protein (20-60 μg) was mixed with an equalvolume of 2× sample buffer (0.125M Tris, pH 6.8, 4% SDS, 20% glycerol,20% 2-mercaptoethanol), boiled for 5 min, and electrophoresed on a 7.5%SDS-PAGE gel, and transferred to a nitrocellulose membrane. The αllbintegrin subunit was detected by mouse mAb MAB 1990 (10 ng/ml, Chemicon,Temecula, Calif.) as described. Trikha, M. et al., Cancer Res.56:5071-5078 (1996). The tr-αllb integrin subunit was detected byprotein G purified pAb MTB1at a dilution of 1:2,000. Bound primaryantibody was detected by species matched secondary lg conjugated to HRP(1:1,500 dilution, Amersham, Arlington Heights, Ill.), and the bandswere visualized by ECL detection (Amersham). Alternatively, pAb MTB1 wasbiotinylated (Amersham) and used as a primary Ab (1:2,000 dilution),followed by detection with Streptavidin conjugated to HRP (1:2,000dilution) and ECL. The peptide antigen (8-fold molar excess) waspreincubated with pAb MTB1 prior to Western blotting in order to competeout binding of the Ab to the membrane. Substitution of primary Ab withpreimmune serum or absence of primary Ab served as negative controls.

SPECIFIC EXAMPLE 4 Results

Immunohistochemical detection of tr-αllb and tr-β3 in prostateadenocarcinoma tissue. The results described above indicate that tr-αllband tr-β3 integrins are expressed in cultured tumor cell lines. Toconfirm that expression of these integrins was not an artifact of invitro cell culture, immunohistochemical staining of radicalprostatectomy specimens obtained from patients who had undergone surgeryfor localized prostate adenocarcinoma was performed. Specimens from fivepatients indicated specific staining of pAb MTB1 and pAb MTB4 (describedin the Methods of Specific Example 2) in prostate adenocarcinoma cells(brown color, FIGS. 7A, 7C). In normal prostatic tissue, pAb MTB1 andpAb MTB4 showed no immunoreactivity. Substitution of pAb MTB1 and pAbMTB4 with pre-immune serum (FIGS. 7B, 7D), served as a negative control.Results from these studies indicate that tr-αllb and tr-β3 are not justexpressed in cultured tumor cell lines, but are also expressed inprostate adenocarcinoma tumor tissue.

Methods

Immunohistochemistry. Representative formalin-fixed paraffin-embeddedtissue sections were selected from radical prostatectomy specimens ofpatients who had undergone surgery for clinically localizedadenocarcinoma of the prostate. All specimens were handled according toa previously described protocol. Sakr, W. A. et al., J. Urol. Path.3:355-364 (1995). Sections were chosen to include normal and neoplastictissue. The ABC method (Vector laboratories, Burlingame, Calif.) wasused to visualize the binding of pAb MTB1, pAb MTB4 and the preimmuneserum. Briefly, deparaffinized and blocked specimens were incubated withpAb MTB1 or pAb MTB4, or the pre-immune serum at a dilution of 1:100 for1 h at room temperature. Following incubation, sections were washed andincubated with biotinylated anti-rabbit Ab (1:200 dilution) for 10 min,washed, and incubated with avidin-biotin complex peroxidase reagent andsubstrate for another 10 min. After color development the sections werecounterstained with hematoxylin.

SPECIFIC EXAMPLE 5

Patients with metastatic disease may secrete the truncated integrinseither locally or into the blood, serum or urine. The secreted proteinscould thus be detected by an enzyme linked immunosorbent assay (ELISA),dot, and/or Western blotting techniques. In an ELISA, proteins areimmobilized on 96-well ELISA plates, followed by the addition ofantibody, e.g., pAb MTB1 or pAb MTB4, and goat anti-rabbit Ig antibodyconjugated to Horse Radish Peroxidase (HRP). After the addition of theHRP-substrate, extent of color development is monitored by an ELISAplate reader. Trikha, M. et al., Cancer Res. 54:4993-4998 (1994). Thisapproach quantitates the level of wild type- and tr-integrins which isthen compared with stage and outcome of disease.

Expression of wild type and tr-integrin mRNAs in tumor specimens by Insitu hybridization (ISH), RACE, and immunohistochemistry can also bedetermined. For ISH, sense and antisense riboprobes specific to thetr-integrin and the wild type integrin mRNAs are provided.Alternatively, a quantitative RT-PCR is used to compare the relativeamount of gene expression of tr-integrin mRNAs in tumor versus normaltissue.

tr-integrins of the present invention may act as endogenous disintegrinsand thereby confer a metastatic potential on tumor cells. The secretedintegrin receptor could bind to extracellular matrix proteins andcompete for binding with cell surface associated integrins. Sincemetastatic progression involves a change in the adhesive phenotype oftumor cells (Tang, D. G. et al., Invasion Metastasis 95:109-122 (1994)),expression of truncated integrins could make the tumor cells lessadhesive and possibly more metastatic. Antimetastatic therapy based ondrugs that neutralize the effect of integrins is thus also encompassedby the present invention. For example, such drugs could interfere withintegrin function by inhibiting binding of the in vivo substrate to theintegrin receptor and/or inhibiting association of the integrin subunitsto form a functional receptor. These drugs could also target geneexpression of the truncated integrins.

The foregoing discussion discloses and describes merely exemplaryembodiments of the present invention. One skilled in the art willreadily recognize from such discussion, and from the accompanyingdrawings and claims, that various changes, modifications and variationscan be made therein without departing from the spirit and scope of theinvention as defined in the following claims.

All patents and other publications cited herein are expresslyincorporated by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                  - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 9                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 753 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - GCAGATACGG AGCAAGAACA GCCAGAATCC AAACAGCAAG ATTGTGCTGC TG -             #GACGTGCC     60                                                                 - - GGTCCGGGCA GAGGCCCAAG TGGAGCTGCG AGGGAACTCC TTTCCAGCCT CC -            #CTGGTGGT    120                                                                 - - GGCAGCAGAA GAAGGTGAGA GGGAGCAGAA CAGCTTGGAC AGCTGGGGAC CC -            #AAAGTGGA    180                                                                 - - GCACACCTAT GAGCTCCACA ACAATGGCCC TGGGACTGTG AATGGTCTTC AC -            #CTCAGCAT    240                                                                 - - CCACCTTCCG GGACAGTCCC AGCCCTCCGA CCTGCTCTAC ATCCTGGATA TA -            #CAGCCCCA    300                                                                 - - GGGGGGCCTT CAGTGCTTCC CACAGCCTCC TGTCAACCCT CTCAAGGTGG AC -            #TGGGGGCT    360                                                                 - - GCCCATCCCC AGCCCCTCCC CCATTCACCC GGCCCATCAC AAGCGGGATC GC -            #AGACAGAT    420                                                                 - - CTTCCTGCCA GAGCCCGAGC AGCCCTCGAG GCTTCAGGAT CCAGTTCTCG TA -            #GTGAGCAG    480                                                                 - - GCTCTCTGGT CTCTGGCCCG GCCTCCCCGG GACCCACGGG GCAGAGGGGA TG -            #GGAGGAGG    540                                                                 - - GAGAGGGGTC CGGGTGTGCT GTGGGCCTCT GTGGGCCACG CTTGGTCCCT GG -            #GAGCACTT    600                                                                 - - CAAGTGAACA TGGAGGAGCA TGCTGGCTTG TGTCTGGGGT GAGCTGAAAG AC -            #ACTTGCAC    660                                                                 - - TTTTTAAAAG CTTCCCAGTA CGTTAAGGAG CATAAAACAA TGCCAAAGCA AG -            #GTTAAAAA    720                                                                 - - AAAAAAAAAA AAAGTACTAG TCGACGCGTG GCC       - #                  -      #        753                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 201 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Gln Ile Arg Ser Lys Asn Ser Gln Asn Pro As - #n Ser Lys Ile Val        Leu                                                                             1               5   - #                10  - #                15              - - Leu Asp Val Pro Val Arg Ala Glu Ala Gln Va - #l Glu Leu Arg Gly Asn                  20      - #            25      - #            30                   - - Ser Phe Pro Ala Ser Leu Val Val Ala Ala Gl - #u Glu Gly Glu Arg Glu              35          - #        40          - #        45                       - - Gln Asn Ser Leu Asp Ser Trp Gly Pro Lys Va - #l Glu His Thr Tyr Glu          50              - #    55              - #    60                           - - Leu His Asn Asn Gly Pro Gly Thr Val Asn Gl - #y Leu His Leu Ser Ile      65                  - #70                  - #75                  - #80        - - His Leu Pro Gly Gln Ser Gln Pro Ser Asp Le - #u Leu Tyr Ile Leu Asp                      85  - #                90  - #                95               - - Ile Gln Pro Gln Gly Gly Leu Gln Cys Phe Pr - #o Gln Pro Pro Val Asn                  100      - #           105      - #           110                  - - Pro Leu Lys Val Asp Trp Gly Leu Pro Ile Pr - #o Ser Pro Ser Pro Ile              115          - #       120          - #       125                      - - His Pro Ala His His Lys Arg Asp Arg Arg Gl - #n Ile Phe Leu Pro Glu          130              - #   135              - #   140                          - - Pro Glu Gln Pro Ser Arg Leu Gln Asp Pro Va - #l Leu Val Val Ser Arg      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Leu Ser Gly Leu Trp Pro Gly Leu Pro Gly Th - #r His Gly Ala Glu        Gly                                                                                             165  - #               170  - #               175             - - Met Gly Gly Gly Arg Gly Val Arg Val Cys Cy - #s Gly Pro Leu Trp Ala                  180      - #           185      - #           190                  - - Thr Leu Gly Pro Trp Glu His Phe Lys                                              195          - #       200                                             - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 37 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - GGCCACGCGT CGACTAGTAC TTTTTTTTTT TTTTTTT      - #                       - #      37                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - CTGGAAGAGG CTGGGAGTC             - #                  - #                      - # 19                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - GGCCACGCGT CGACTAGTAC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - GCAGATACGG AGCAAGAACA            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - Thr His Gly Ala Glu Gly Met Gly Gly Gly Ar - #g Gly Val Arg Val Cys      1               5   - #                10  - #                15               - - Cys Gly Pro Leu Trp Ala Thr Leu Gly Pro Tr - #p Glu His Phe Lys                      20      - #            25      - #            30                   - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS: unknown                                                     (D) TOPOLOGY: unknown                                                - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - Cys Pro Gly Ala Ser Val Gly Thr Gly Pro Pr - #o Phe Phe Leu Leu          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -    (iii) HYPOTHETICAL: NO                                                 - -     (iv) ANTI-SENSE: NO                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - GCCTCTAGAG CCACCATGGC CAGAGCTTTG TG       - #                  - #              32                                                                    __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule encoding tr-αllb,consisting of the nucleotide sequence of SEQ ID NO: 1.